Background <p>Reuse of plant growing substrate can contribute to lowering the carbon footprint of horticulture production systems. Here, we assessed the impact of substrate reusing on the root-associated microbiome of strawberries. The cultivars Elsanta and Malling Centenary were grown in a substrate-based hydroponic system using either fresh peat-based substrate or substrate reused up to three times, with comparisons made between not steamed and steam-treated substrate. The root-associated microbiome was analyzed using 16S rRNA gene and ITS1 DNA sequencing to determine bacterial and fungal communities.</p> Results <p>Substrate reusing without steaming increased the bacterial and fungal community diversity whereas steaming reduced the bacterial diversity and increased fungal diversity in the root-associated microbiome. The root-associated bacterial communities recruited by the two cultivars were diverse, even more so than the diversity recorded for the different times of reused substrate.</p> Conclusion <p>These observations demonstrate the ability of strawberry to establish a genotype-specific root-associated microbiome when plants are cultured on reused substrate. The bacterial microbiome showed a higher consistency over the times substrate was reused, while the fungal community composition showed stronger adaptation to the substrate reusing. Pathogenic fungi accumulated over the reusing times, underscoring the necessity of substrate sanitation through steaming to minimize the risk of pathogen infections.</p> Clinical trial number <p>Not applicable.</p>